GSM6809578: CD200R_Ear, Replicate 1, mRNA-derived cDNA; Mus musculus; OTHER
Both ears from groups of 2-3 mice within the same treatment group were pooled together into two biological replicates for digestion using the Multi Tissue Dissociation Kit 1 (Milenyi) according to manufacturer's recommendations in the Dissociation of mouse Ear protocol. The resulting single cell suspensions were incubated in Cell Staining Buffer (BioLegend) containing Human TruStain FcX (BioLegend) on ice for 15 minutes protected from light prior to addition of Live/Dead Fixable Near-IR (Invitrogen), AlexaFluor700-conjugated anti-human CD45 (clone 2D1, eBioscience), anti-human CD200R (clone OX-108, BioLegend) that was previously biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin kit (ThermoFisher), and TotalSeq-B conjugated anti-human antibodies (CD20, CD3, CD33, CD4, CD8a, HLA-DR, and CD123, BioLegend) for 30 minutes on ice, protected from light. Cells were washed twice in Cell Staining Buffer prior to incubation with TotalSeq-B-PE-conjugated Streptavidin (BioLegend) diluted in Cell Staining Buffer for 20 minutes on ice and washed twice in Cell Staining Buffer. Cells were diluted in Cell Staining Buffer to 2x10^7 cells/mL (500 uL minimum volume) and collected in RPMI (ThermoFisher) supplemented with 50% FBS (ThermoFisher) using a FACSAria III cell sorter (BD Biosciences). Following cell sorting, if yields were higher than 20, 000 cells, cells were counted and diluted to 1x10^6 cells/mL in DPBS and 10, 000 cells from each sample were loaded onto separate lanes of a Chromium Controller instrument (10x Genomics). For samples with yields lower than 20, 000 cells, the entire sample was loaded onto an individual lane of a Chromium Controller. The resulting GEMs were processed according to the standard 10x Genomics single cell 3' v3.1 assay protocol. After library preparation and i7 indexing of individual samples, the final pooled library was comprised of 90% mRNA-derived library and 10% ADT library.